Eudistomin Y-type covalent derivatives with antitumor activity, their preparation methods and applications

Abstract

This invention discloses Eudistomin Y-type covalent derivatives of formula (I) and their medically acceptable salts, as well as methods for their preparation. The Eudistomin Y-type covalent derivatives and their medically acceptable salts provided by this invention possess antitumor activity, and their covalent groups can bind to target proteins in cells, resulting in a longer duration of action and a sustained antitumor effect, which can be used to prepare long-acting antitumor drugs.

Description

Technical Field

[0001] This invention relates to a class of novel Eudistomin Y covalent derivatives, their preparation methods, and their application as antitumor drugs, belonging to the field of pharmaceutical technology. Background Technology

[0002] Triple-negative breast cancer, lacking known drug targets, has the worst prognosis among breast cancers, is prone to metastasis, and has a high recurrence rate. The design and research of innovative anti-tumor drugs targeting triple-negative breast cancer still face significant challenges, and the development of innovative anti-tumor drugs remains crucial.

[0003] Marine-derived natural products are an important source for new drug development. Eudistomin Y-class compounds, a class of β-carboline marine alkaloids, were isolated and identified from Eudistominoma sea squirts in 2008 and represent a novel type of marine alkaloid. Our previous studies found that Eudistomin Y-class derivatives possess activity in reversing multidrug resistance in tumors (patent application publication number CN113651815A), and also discovered that their derivatives have good antitumor activity (patent application publication number CN111423438A). However, due to the lack of a strong interaction between the drug and the potential target, it is difficult to produce a relatively stronger effect. This invention aims to construct novel covalent compounds of Eudistomin Y-class by introducing covalently reactive modifying groups to obtain new compounds with good antitumor activity against triple-negative breast cancer. Summary of the Invention

[0004] To design novel covalent derivatives of the Eudistomin Y class with excellent antitumor activity and convenient preparation, and to further provide a drug and composition thereof for treating corresponding diseases or symptoms, this invention provides a series of derivatives with the structural characteristics of general formula (I) and their pharmaceutically acceptable salts. The antitumor activity of these compounds is discovered for the first time, and the compounds are synthesized for the first time.

[0005] The first objective of this invention is to provide a Eudistomin Y-type covalent derivative with good antitumor activity.

[0006] To achieve the objective of this invention, the technical solution adopted is as follows:

[0007] Formula (I) shows Eudistomin Y-type covalent derivatives and their medically acceptable salts.

[0008]

[0009] (I)

[0010] in,

[0011] R1 represents methyl, ethyl, n-butyl, cyclopropylmethyl, cyclohexylmethyl, (tetrahydropyran-4-yl)methyl or 4-methoxybenzyl, and R2 represents 2,3-epoxypropyl or acetonitrile.

[0012] Furthermore, when R2 is preferably 2,3-epoxypropyl, R1 is methyl, ethyl, n-butyl, cyclopropylmethyl, cyclohexylmethyl, (tetrahydropyran-4-yl)methyl or 4-methoxybenzyl.

[0013] Or, if R2 is acetonitrile, R1 is cyclohexylmethyl or 4-methoxybenzyl;

[0014] More preferably, R1 is methyl, ethyl or (tetrahydropyran-4-yl)methyl, and R2 is 2,3-epoxypropyl;

[0015] Alternatively, R1 may be cyclohexylmethyl and R2 may be acetonitrile.

[0016] The preferred formula (I) shows one of the following covalent derivatives of Eudistomin Y:

[0017]

[0018] A second objective of this invention is to provide a method for preparing Eudistomin Y-type covalent derivatives, the reaction formula of which is as follows:

[0019]

[0020] The definitions of R1 and R2 are as described above.

[0021] The method includes the following steps:

[0022] (1) Using tryptophan methyl ester as a raw material, it is reacted with 4-methoxyacetophenone, I2 and H2O2 in a molar ratio of 1:1~1.5:0.8~2:1.5~2.5 to obtain the intermediate compound shown in Formula 1;

[0023] The reaction solvent in step (1) is an aprotic solvent; preferably DMSO.

[0024] The reaction temperature in step (1) is 100℃-150℃; preferably 100℃-130℃.

[0025] The reaction time for step (1) is 3-8 hours; preferably 5-7 hours. The reaction is typically monitored by TLC until completion.

[0026] (2) The intermediate compound shown in Formula 1 is reacted with R1-X and K2CO3 in a molar ratio of 1:1.7 to 6:1.2 to 7 to obtain the compound shown in Formula 2;

[0027] In R1-X, the X group is a halogen element;

[0028] Preferably, X is Cl, Br, or I.

[0029] The reaction solvent in step (2) is an aprotic solvent; preferably anhydrous DMF.

[0030] The reaction temperature in step (2) is -20℃ to 100℃; preferably -5℃ to 30℃.

[0031] Preferably, the reaction solvent is anhydrous DMF; the reaction temperature is -5℃ to 30℃.

[0032] The reaction time for step (2) is 12 to 24 hours; preferably 12 to 20 hours. The reaction is typically monitored by TLC until completion.

[0033] (3) The compound shown in Formula 2 is hydrolyzed under alkaline conditions to obtain the compound shown in Formula 3;

[0034] The reaction conditions in step (3) are an alkaline environment with a pH of 8-13, preferably 9-12. NaOH is typically added to adjust the pH to 8-13.

[0035] The reaction solvent in step (3) is preferably a proton solvent, such as methanol.

[0036] The reaction temperature in step (3) is 50-80℃; preferably, the reaction temperature is 50-60℃.

[0037] The reaction time for step (3) is 6-10 hours, preferably 8-10 hours. The reaction is typically monitored by TLC until it is complete.

[0038] (4) The compound shown in Formula 3 was reacted with R2-Y and K2CO3 in a molar ratio of 1:1.2 to 10:1.5 to 8 to prepare Eudistomin Y-type covalent derivatives as shown in Formula (I);

[0039] In R2-Y, the Y group is a halogen element;

[0040] Preferably, Y is Cl, Br, or I.

[0041] The reaction solvent in step (4) is an aprotic solvent, preferably anhydrous DMF;

[0042] The reaction temperature in step (4) is -20℃ to 100℃; preferably 0-30℃.

[0043] The reaction time for step (4) is 10 to 24 hours; preferably 12 to 18 hours. The reaction is typically monitored by TLC until completion.

[0044] A third objective of this invention is to provide the application of Eudistomin Y covalent derivatives and their medically acceptable salts in the preparation of antitumor drugs. Further, it is preferred that the antitumor drug be used to treat triple-negative breast cancer, but it is not limited to the treatment and prevention of triple-negative breast cancer. The Eudistomin Y covalent derivatives of formula (I) of this invention possess antitumor activity, particularly exhibiting good antiproliferative activity against triple-negative breast cancer MDA-MB-231 cells.

[0045] Furthermore, the Eudistomin Y covalent derivatives shown in formula (I) of this invention can bind to target proteins in cells due to their covalent groups, thus acting on tumor cells for a longer period of time in vivo. They are not easily expelled by cells, resulting in a longer duration of action and a long-lasting anti-tumor effect.

[0046] Therefore, this invention provides the application of Eudistomin Y covalent derivatives and their medically acceptable salts in the preparation of long-acting antitumor drugs.

[0047] The fourth objective of this invention is to provide an application of Eudistomin Y covalent derivatives and their medically acceptable salts in the preparation of antitumor tracer molecular tool drugs. The Eudistomin Y covalent derivatives shown in formula (I) are fluorescent compounds with fluorescent properties, which can effectively trace the distribution of the compound in cells and tissues through fluorescence characteristics.

[0048] The pharmaceutically acceptable salts described in this invention refer to conventional acid addition salts that have the same pharmaceutical efficacy as the derivatives and are formed with suitable non-toxic organic or inorganic acids.

[0049] The present invention also discloses a pharmaceutical composition comprising a covalent derivative of Eudistomin Y class as shown in formula (I) of the present invention or a pharmaceutically acceptable salt thereof, which can be added with a pharmaceutically acceptable carrier to form common pharmaceutical preparations such as tablets, capsules, powders, syrups, liquids, suspensions, and injections, and can be added with commonly used pharmaceutical excipients such as flavorings, sweeteners, liquid or solid fillers or diluents.

[0050] The pharmaceutical composition described in this invention can be administered clinically via oral administration, injection, or other methods.

[0051] The clinical dosage of the compounds of this invention is 0.01 mg to 1000 mg / day, but may deviate from this range depending on the severity of the condition or the dosage form.

[0052] The present invention has the following advantages over the prior art:

[0053] The Eudistomin Y-type covalent derivatives shown in this invention have good antitumor activity, can be effectively fluorescently traced, and can bind to multiple targets, producing a strong and long-lasting effect on the corresponding targets, which can effectively reduce the occurrence of drug resistance. Attached Figure Description

[0054] Figure 1 Bar graph showing the activity of compounds 4-12 in inhibiting the proliferation of triple-negative breast cancer MDA-MB-231 cells.

[0055] Figure 2 This is a protein gel electrophoresis fluorescence image of the covalently bound target protein of compound 4 in MDA-MB-231 cells.

[0056] Figure 3 The images show the fluorescence of compounds 4, 5, 9, and 10 in MDA-MB-231 cells. Detailed Implementation

[0057] The technical solution of the present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to these embodiments.

[0058] Example 1

[0059] Preparation of compound 4

[0060]

[0061] In a 100 mL round-bottom flask, L-tryptophan methyl ester, 4-methoxyacetophenone (417 mg), and elemental iodine (610 mg) were dissolved in dimethyl sulfoxide (7 mL). 30% hydrogen peroxide (1.5 eq) was slowly added dropwise at 0 °C, and the mixture was stirred at room temperature for about 20 minutes. The mixture was then transferred to an oil bath at 110 °C, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, the solution temperature was lowered to room temperature, and deionized water, ethyl acetate, saturated ammonium chloride solution, and 10% sodium thiosulfate solution were added sequentially for extraction. Anhydrous sodium sulfate was added to the organic phase and dried for 15–20 minutes. The mixture was filtered, concentrated, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate) to obtain target compound 1. Under argon protection, in a 25 mL round-bottom flask, 1 (30 mg) was dissolved in dry DMF (1 mL), potassium carbonate (69 mg) was added, and iodomethane (83 mg) was slowly added under an ice-water bath. The mixture was stirred in the dark at room temperature, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, deionized water was added to quench the reaction, and the mixture was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic phase and dried for 15-20 minutes. The mixture was filtered, concentrated, and subjected to silica gel column chromatography (200-300 mesh) (eluent: petroleum ether / ethyl acetate) to obtain intermediate compound 4-2. Intermediate compound 4-2 was dissolved in a methanol solution of sodium hydroxide, the pH was adjusted to 11, and the mixture was hydrolyzed at 50-60 °C. The reaction was monitored by thin-layer chromatography. After the hydrolysis was completed, the mixture was concentrated to obtain intermediate compound 4-3. Under argon protection, 25 mg of compound 4-3 was dissolved in dry DMF (1 mL), potassium carbonate (64 mg) was added, and 66 μL of epoxybromopropane was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by TLC until completion. After quenching with deionized water, the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate for 15-20 minutes, filtered, concentrated, and purified by silica gel column chromatography (200-300 mesh) to obtain the target compound 4.

[0062] 1-(4-methoxy)benzoyl)-3-(2,3)-glycidyl-9-methyl-β-carboline(4); 1H NMR (400MHz, CDCl3) δ8.93 (s, 1H), 8.19 (d, J = 7.8Hz, 1H), 8.02 (d, J = 9.0Hz, 2H), 7.61 (dd d,J=8.3,7.2,1.2Hz,1H),7.45(d,J=8.4Hz,1H),7.34(ddd,J=8.0,7.2,0.9Hz,1H),6.91( d,J=9.0Hz,2H),4.68(dd,J=12.3,3.2Hz,1H),4.24(dd,J=12.3,5.9Hz,1H),3.82(s,3H), 3.73(s,3H),3.37–3.32(m,1H),2.83(dd,J=4.9,4.1Hz,1H),2.72(dd,J=4.9,2.6Hz,1H).

[0063] Example 2

[0064] Preparation of compound 5

[0065]

[0066] Under argon protection, in a 25 mL round-bottom flask, 1 (30 mg) was dissolved in dry DMF (1 mL), potassium carbonate (86 mg) was added, and bromoethane (37 μL) was slowly added under an ice-water bath. The mixture was stirred in the dark at room temperature, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, deionized water was added to quench the reaction, and the mixture was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic phase and dried for 15–20 minutes. The mixture was filtered, concentrated, and subjected to silica gel column chromatography (200–300 mesh) (eluent: petroleum ether / ethyl acetate) to obtain intermediate compound 5-2. Intermediate compound 5-2 was dissolved in a methanol solution of sodium hydroxide, the pH was adjusted to 11, and the mixture was hydrolyzed at 50–60 °C. The reaction was monitored by thin-layer chromatography. After the hydrolysis was completed, the mixture was concentrated to obtain intermediate compound 5-3. Under argon protection, 25 mg of compound 5-3 was dissolved in dry DMF (1 mL), potassium carbonate (70 mg) was added, and 64 μL of epoxide was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by TLC until completion. After quenching with deionized water, the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate for 15-20 minutes, filtered, concentrated, and purified by silica gel column chromatography (200-300 mesh) to obtain the target compound 5.

[0067] 1-(4-(methoxy)benzoyl)-3-(2,3)-glycidyl-9-ethyl-β-carboline (5); 1H NMR (400MHz, CDCl3) δ9.00 (s, 1H), 8.27 (d, J = 7.9Hz, 1H), 8.04 (d, J = 9.0Hz, 2H), 7.68 (dd, J = 15 .5,1.2Hz,1H),7.54(d,J=8.4Hz,1H),7.41(dd,J=15.0,0.8Hz,1H),6.98(d,J=9.0Hz,2H),4.75 (dd,J=12.3,3.2Hz,1H),4.37(q,J=7.2Hz,2H),4.31(dd,J=12.3,5.9Hz,1H),3.89(s,3H),3.4 5–3.38(m,1H),2.89(dd,J=4.9,4.1Hz,1H),2.78(dd,J=4.9,2.6Hz,1H),1.24(t,J=7.2Hz,3H).

[0068] Example 3

[0069] Preparation of compound 6

[0070]

[0071] Under argon protection, in a 25 mL round-bottom flask, 1 (30 mg) was dissolved in dry DMF (1 mL), potassium carbonate (77 mg) was added, and bromobutane (54 μL) was slowly added under an ice-water bath. The mixture was stirred in the dark at room temperature, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, deionized water was added to quench the reaction, and the mixture was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic phase and dried for 15–20 minutes. The mixture was filtered, concentrated, and subjected to silica gel column chromatography (200–300 mesh) (eluent: petroleum ether / ethyl acetate) to obtain intermediate compound 6-2. Intermediate compound 6-2 was dissolved in a methanol solution of sodium hydroxide, the pH was adjusted to 11, and the mixture was hydrolyzed at 50–60 °C. The reaction was monitored by thin-layer chromatography. After the hydrolysis was completed, the mixture was concentrated to obtain intermediate compound 6-3. Under argon protection, 25 mg of 6-3 was dissolved in dry DMF (1 mL), potassium carbonate (74 mg) was added, and epichlorohydrin (59 μL) was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by TLC until completion. After quenching with deionized water, the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate for 15-20 minutes, filtered, concentrated, and purified by silica gel column chromatography (200-300 mesh) to obtain the target compound 6.

[0072] 1-(4-(methoxy)benzoyl)-3-(2,3)-epoxypropyl-9-n-butyl-β-carboline(6); 1HNMR(400MHz, CDCl3) δ9.00(s,1H),8.26(d,J=7.9Hz,1H),8.05(d,J=9.0Hz,2H),7.66(ddd,J=8.4,7 .2,1.2Hz,1H),7.52(d,J=8.4Hz,1H),7.40(ddd,J=8.0,7.1,0.9Hz,1H),6.97(d,J=9.0Hz,2H),4.75 (dd,J=12.2,3.2Hz,1H),4.35–4.26(m,3H),3.89(s,3H),3.44–3.39(m,1H),2.89(dd,J=4.9,4.1Hz, 1H), 2.78 (dd, J=4.9, 2.6Hz, 1H), 1.56 (p, J=7.7Hz, 2H), 1.15 (h, J=7.4Hz, 2H), 0.72 (t, J=7.4Hz, 3H).

[0073] Example 4

[0074] Preparation of compound 7

[0075]

[0076] Under argon protection, in a 25 mL round-bottom flask, 1 (30 mg) was dissolved in dry DMF (1 mL), potassium carbonate (79 mg) was added, and bromomethylcyclopropane (50 μL) was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, deionized water was added to quench the reaction, and the mixture was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic phase and dried for 15-20 minutes. The mixture was filtered, concentrated, and subjected to silica gel column chromatography (200-300 mesh) (eluent: petroleum ether / ethyl acetate) to obtain intermediate compound 7-2. Intermediate compound 7-2 was dissolved in a methanol solution of sodium hydroxide, the pH was adjusted to 11, and the mixture was hydrolyzed at 50-60 °C. The reaction was monitored by thin-layer chromatography. After the hydrolysis was completed, the mixture was concentrated to obtain intermediate compound 7-3. Under argon protection, 25 mg of 7-3 was dissolved in dry DMF (1 mL), potassium carbonate (68 mg) was added, and epichlorohydrin (60 μL) was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by TLC until completion. After quenching with deionized water, the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate for 15-20 minutes, filtered, concentrated, and purified by silica gel column chromatography with a mesh of 200-300 to obtain the target compound 7.

[0077] 1-(4-(methoxy)benzoyl)-3-(2,3)-glycidyl-9-cyclopropylmethyl-β-carboline (7); 1 H NMR (400MHz, CDCl3) 1H NMR (400MHz, CDCl3)δ 1 H NMR(400MHz,Chloroform-d)δ9.02(s,1H),8.27(d,J=7.9Hz,1H),8.08(d,J=9.0Hz,2H),7.66(ddd,J =8.3,7.1,1.2Hz,1H),7.55(d,J=8.4Hz,1H),7.47–7.36(m,1H),6.98(d,J=9.0Hz,2H),4.75(dd,J=1 2.3,3.2Hz,1H),4.36–4.28(m,3H),3.89(s,3H),3.46–3.35(m,1H),2.89(dd,J=4.9,4.2Hz,1H),2.7 9(dd,J=4.9,2.6Hz,1H),1.06–0.95(m,1H),0.29(dt,J=8.1,5.3Hz,2H),0.15(dt,J=6.1,4.8Hz,2H).

[0078] Example 5

[0079] Preparation of compound 8

[0080]

[0081] Under argon protection, in a 25 mL round-bottom flask, 1 (30 mg) was dissolved in dry DMF (1 mL), potassium carbonate (74 mg) was added, and bromomethylcyclohexane (70 μL) was slowly added under an ice-water bath. The mixture was stirred in the dark at room temperature, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, deionized water was added to quench the reaction, and the mixture was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic phase and dried for 15–20 minutes. The mixture was filtered, concentrated, and subjected to silica gel column chromatography (200–300 mesh) (eluent: petroleum ether / ethyl acetate) to obtain intermediate compound 8-2. Intermediate compound 8-2 was dissolved in a methanol solution of sodium hydroxide, the pH was adjusted to 11, and the mixture was hydrolyzed at 50–60 °C. The reaction was monitored by thin-layer chromatography. After the hydrolysis was completed, the mixture was concentrated to obtain intermediate compound 8-3. Under argon protection, 25 mg of 8-3 was dissolved in dry DMF (1 mL), potassium carbonate (70 mg) was added, and 66 μL of epichlorohydrin was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by TLC until completion. After quenching with deionized water, the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate for 15-20 minutes, filtered, concentrated, and purified by silica gel column chromatography (200-300 mesh) to obtain the target compound 8.

[0082] 1-(4-(methoxy)benzoyl)-3-(2,3)-glycidyl-9-cyclohexylmethyl-β-carboline(8); 1H NMR(400MHz, CDCl3)δ9.01(s,1H),8.26(d,J=7.6Hz,1H),8.09(d,J=9.0Hz,2H),7.65(ddd,J=8.4,7.1,1.2Hz,1H), 7.54(d,J=8.4Hz,1H),7.39(ddd,J=7.9,7.1,0.8Hz,1H),6.98(d,J=9.0Hz,2H),4.76(dd,J=12.3,3.2Hz,1H),4.32( dd,J=12.3,5.8Hz,1H),4.19(d,J=7.2Hz,2H),3.90(s,3H),3.44–3.39(m,1H),2.89(dd,J=5.0,4.1Hz,1H),2.79(dd ,J=4.9,2.6Hz,1H),1.50–1.40(m,4H),1.25–1.20(m,2H),1.02–0.89(m,1H),0.89–0.76(m,2H),0.72–0.59(m,2H).

[0083] Example 6

[0084] Preparation of compound 9

[0085]

[0086] Under argon protection, in a 25 mL round-bottom flask, 1 (30 mg) was dissolved in dry DMF (1 mL), potassium carbonate (83 mg) was added, and bromomethylcyclohexane (70 μL) was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, deionized water was added to quench the reaction, and the mixture was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic phase and dried for 15-20 minutes. The mixture was filtered, concentrated, and subjected to silica gel column chromatography (200-300 mesh) (eluent: petroleum ether / ethyl acetate) to obtain intermediate compound 9-2. Intermediate compound 9-2 was dissolved in a methanol solution of sodium hydroxide, the pH was adjusted to 11, and the mixture was hydrolyzed at 50-60 °C. The reaction was monitored by thin-layer chromatography. After the hydrolysis was completed, the mixture was concentrated to obtain intermediate compound 9-3. Under argon protection, 25 mg of 9-3 was dissolved in dry DMF (1 mL), potassium carbonate (75 mg) was added, and bromoacetonitrile (72 μL) was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by TLC until completion. After quenching with deionized water, the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate for 15-20 minutes, filtered, concentrated, and purified by silica gel column chromatography (200-300 mesh) to obtain the target compound 9.

[0087] 1-(4-(methoxy)benzoyl)-3-acetonitrile-9-cyclohexylmethyl-β-carbamate (9);1 H NMR (400MHz, CDCl3) δ9.04 (s, 1H), 8.26 (d, J = 7.9Hz, 1H), 8.05 (d, J = 9.0Hz, 2H), 7.67(ddd,J=8.3,7.1,1.2Hz,1H),7.55(d,J=8.4Hz,1H),7.41(ddd,J=8.0,7.1, 0.9Hz,1H),6.98(d,J=9.0Hz,2H),5.04(s,2H),4.18(d,J=7.2Hz,2H),3.91(s,3 H),1.52–1.40(m,4H),1.26–1.20(m,2H),1.04–0.75(m,3H),0.72–0.58(m,2H).

[0088] Example 7

[0089] Preparation of compound 10

[0090]

[0091] Under argon protection, in a 25 mL round-bottom flask, 1 (30 mg) was dissolved in dry DMF (1 mL), potassium carbonate (96 mg) was added, and 4-bromomethyltetrahydropyran (89 mg) was slowly added under an ice-water bath. The mixture was stirred in the dark at room temperature, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, deionized water was added to quench the reaction, and the mixture was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic phase and dried for 15-20 minutes. The mixture was filtered, concentrated, and subjected to silica gel column chromatography (200-300 mesh) (eluent: petroleum ether / ethyl acetate) to obtain intermediate compound 10-2. Intermediate compound 10-2 was dissolved in a methanol solution of sodium hydroxide, the pH was adjusted to 11, and the mixture was hydrolyzed at 50-60 °C. The reaction was monitored by thin-layer chromatography. After the hydrolysis was completed, the mixture was concentrated to obtain intermediate compound 10-3. Under argon protection, 25 mg of 10⁻³ was dissolved in dry DMF (1 mL), potassium carbonate (68 mg) was added, and 64 μL of epichlorohydrin was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by TLC until completion. After quenching with deionized water, the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate for 15-20 minutes, filtered, concentrated, and purified by silica gel column chromatography (200-300 mesh) to obtain the target compound 10.

[0092] 1-(4-(methoxy)benzoyl)-3-(2,3)-glycidyl-9-(tetrahydropyran-4-yl)methyl-β-carboline (10); 1H NMR (400MHz, CDCl3) δ9.02(s,1H),8.26(d,J=7.9Hz,1H),8.08(d,J=9.1Hz,2H),7.67(ddd,J=8.4,7.1,1.2Hz,1 H),7.54(d,J=8.5Hz,1H),7.40(ddd,J=7.9,7.1,0.8Hz,1H),6.98(d,J=9.1Hz,2H),4.77(dd,J=12.3,3.1Hz,1H ),4.31(dd,J=12.3,5.9Hz,1H),4.25(d,J=7.2Hz,2H),3.90(s,3H),3.71(dd,J=11.4,3.1Hz,2H),3.44–3.38(m ,1H),2.89(dd,J=4.9,4.2Hz,1H),2.84–2.76(m,3H),1.81–1.73(m,1H),1.30–1.21(m,2H),1.17–1.08(m,2H).

[0093] Example 8

[0094] Preparation of compound 11

[0095]

[0096] Under argon protection, in a 25 mL round-bottom flask, 1 (30 mg) was dissolved in dry DMF (1 mL), potassium carbonate (85 mg) was added, and 4-methoxybenzyl chloride (68 μL) was slowly added under an ice-water bath. The mixture was stirred in the dark at room temperature, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, deionized water was added to quench the reaction, and the mixture was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic phase and dried for 15–20 minutes. The mixture was filtered, concentrated, and subjected to silica gel column chromatography (200–300 mesh) (eluent: petroleum ether / ethyl acetate) to obtain intermediate compound 11-2. Intermediate compound 11-2 was dissolved in a methanol solution of sodium hydroxide, the pH was adjusted to 11, and the mixture was hydrolyzed at 50–60 °C. The reaction was monitored by thin-layer chromatography. After the hydrolysis was completed, the mixture was concentrated to obtain intermediate compound 11-3. Under argon protection, 25 mg of 11-3 was dissolved in dry DMF (1 mL), potassium carbonate (70 mg) was added, and 64 μL of epoxybromopropane was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by TLC until completion. After quenching with deionized water, the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate for 15-20 minutes, filtered, concentrated, and purified by silica gel column chromatography with a mesh of 200-300 to obtain the target compound 11.

[0097] 1-(4-(methoxy)benzoyl)-3-(2,3)-epoxypropyl-9-(4-methoxybenzyl)-β-carbamoline (11); 1 H NMR(400MHz, CDCl3)δ9.02(s,1H),8.30(d,J=7.5Hz,1H),7.70–7.65(m,3H),7.60(d,J=8.4Hz,1H), 7.44(ddd,J=8.0,7.1,0.9Hz,1H),6.77(d,J=9.1Hz,2H),6.55(d,J=8.9Hz,2H),6.35(d,J=8.8Hz,2H ),5.56(s,2H),4.73(dd,J=12.3,3.1Hz,1H),4.28(dd,J=12.3,5.8Hz,1H),3.84(s,3H),3.55(s,3H ), 3.38(dddd,J=5.8,4.1,3.2,2.6Hz,1H),2.86(dd,J=4.9,4.1Hz,1H),2.76(dd,J=4.9,2.6Hz,1H).

[0098] Example 9

[0099] Preparation of compound 12

[0100]

[0101] Under argon protection, in a 25 mL round-bottom flask, 1 (30 mg) was dissolved in dry DMF (1 mL), potassium carbonate (78 mg) was added, and 4-methoxybenzyl chloride (68 μL) was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by thin-layer chromatography. After the reaction was completed, deionized water was added to quench the reaction, and the mixture was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the organic phase and dried for 15-20 minutes. The mixture was filtered, concentrated, and subjected to silica gel column chromatography at 200-300 μM (eluent: petroleum ether / ethyl acetate) to obtain intermediate compound 12-2. Intermediate compound 12-2 was dissolved in a methanol solution of sodium hydroxide, the pH was adjusted to 11, and the mixture was hydrolyzed at 50-60 °C. The reaction was monitored by thin-layer chromatography. After the hydrolysis was completed, the mixture was concentrated to obtain intermediate compound 12-3. Under argon protection, 25 mg of 12-3 was dissolved in dry DMF (1 mL), potassium carbonate (70 mg) was added, and bromoacetonitrile (64 μL) was slowly added at room temperature. The mixture was stirred in the dark at room temperature, and the reaction was monitored by TLC until completion. After quenching with deionized water, the mixture was extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate for 15-20 minutes, filtered, concentrated, and purified by silica gel column chromatography (200-300 mesh) to obtain the target compound 12.

[0102] 1-(4-(methoxy)benzoyl)-3-acetonitrile-9-(methoxybenzyl)-β-carbamate (12); 1 HNMR(400MHz, CDCl3)δ9.05(s,1H),8.31(d,J=7.9Hz,1H),7.70(ddd,J=8.3,7.1,1.2Hz,1H),7.64–7.59(m,3H),7.47(ddd,J=8.0,7.1 ,0.9Hz,1H),6.78(d,J=9.0Hz,2H),6.54(d,J=8.7Hz,2H),6.35(d,J=8.7Hz,2H),5.55(s,2H),5.00(s,2H),3.85(s,3H),3.55(s,3H).

[0103] Example 10: Activity of compound 4-12 against the proliferation of MDA-MB-231 cells

[0104] 1. Experimental Method:

[0105] ① Cell plating: Adherent MDA-MB-231 cells in logarithmic growth phase and in good condition were digested and counted at a concentration of 2.5 × 10⁻⁶. 2 / wells were seeded into 96-well plates and cultured in a 37°C, 5% CO2 cell incubator;

[0106] ② Preparation of drug stock solution: Prepare a 10 mM stock solution of Eudistomin Y covalent derivative using DMSO;

[0107] ③ Cell drug administration: After cells adhered to the plate for 24 hours, 50 μM of different compounds were added for culture, with 3 parallel wells for each group. After drug administration, the 96-well plate was placed in an incubator and incubated statically for 72 hours;

[0108] ④ MTT assay: After 72 h of drug incubation, add 20 μL of 5 mg / mL MTT to each well, incubate at 37 °C and 5% CO2 for 4 h, discard the supernatant, add 150 μL of DMSO to each well, shake for 10 min in the dark to mix, and measure OD570. The group without drug incubation is used as the control group, and the inhibition rate is calculated.

[0109] Inhibition rate = [1 - experimental group (OD570) / control (OD570)] × 100%

[0110] 2. Experimental Results:

[0111] Evaluation analysis of triple-negative breast cancer MDA-MB-231 cells showed that the Eudistomin Y covalent derivatives described in the examples all exhibited good anti-tumor proliferative activity. The results are as follows... Figure 1As shown, the examples exhibited good inhibitory activity against tumor cell proliferation at a concentration of 50 μM.

[0112] The legend is explained below:

[0113] Each value is derived from the mean ± SD of three parallel experiments (n = 3);

[0114] Compared with the control group, #P < 0.05, ##P < 0.01, and ###P < 0.001.

[0115] The above indicates that the Eudistomin Y covalent derivatives shown in general formula (I) exhibit good anti-proliferative activity against MDA-MB-231 cells at a concentration of 50 μM, and their anti-tumor activity is significantly improved compared to the original compound. Compounds 4, 5, 9, and 10 show strong inhibitory activity, with compound 10 achieving an inhibition rate of over 70%.

[0116] Example 11: Fluorescence imaging of target protein labeling of compound 4 in MDA-MB-231 cells

[0117] 1. Experimental Method:

[0118] ① Cell plating: Adherent MDA-MB-231 cells in logarithmic growth phase and in good condition were digested and counted at a concentration of 5 × 10⁻⁶ cells / cells. 5 / wells were seeded into 6-well plates and cultured in a 37°C, 5% CO2 cell incubator;

[0119] ② Preparation of drug stock solution: Prepare a 10 mM stock solution of Eudistomin Y covalent derivative using DMSO;

[0120] ③ Cell drug administration: After the cells adhered to the plate for 24 hours, 10 μM of compound was added for culture. After the drug was added, the 6-well plate was placed in an incubator and cultured statically for 8, 12 and 16 hours.

[0121] ④ SDS-PAGE electrophoresis: After culturing for 16 hours with the drug, the cells were lysed with RIPA lysis buffer for 30 minutes, 5× buffer was added, mixed well, and heated to 100℃ to cook the protein. Then the protein sample was added to the prepared SDS gel wells, and the gel was run at 80V for 30 minutes and at 120V for 1 hour. After electrophoresis, the gel was fluorescently developed.

[0122] 2. Experimental Results:

[0123] Analysis using protein gel electrophoresis fluorescence imaging, such as Figure 2As shown, the prepared Eudistomin Y-type covalent derivative can covalently bind to multiple proteins in MDA-MB-231 cells, and the amount of covalently bound proteins increases with increasing co-incubation time. These results indicate that these proteins are potential target proteins, and the derivative may exert its anti-tumor proliferative effect through covalent binding to these proteins, suggesting that it is a multi-target anti-tumor proliferative drug that can effectively slow down the development of drug resistance.

[0124] Example 12: Fluorescence images of the compound in MDA-MB-231 cells

[0125] 1. Experimental Method:

[0126] ① Cell plating: Adherent MDA-MB-231 cells in logarithmic growth phase and in good condition were digested and counted at a concentration of 1.5 × 10⁻⁶. 4 / wells were seeded in 35mm laser confocal culture dishes and cultured in a 37℃, 5% CO2 cell culture incubator;

[0127] ② Cell drug delivery: After cells adhere to the cell plate for 24 hours, add 40 μM of the compound and incubate in a cell culture incubator for 1 hour.

[0128] ③ Microscopic fluorescence observation: Discard the culture medium, wash three times with PBS, and observe and photograph under a laser confocal microscope. The excitation wavelength is 405nm and the emission wavelength is 465nm.

[0129] 2. Experimental results: Laser confocal microscopy can observe the distribution of compounds in MDA-MB-231 cells and can achieve effective fluorescence tracing.

[0130] Fluorescence images of compounds 4, 5, 9, and 10 in MDA-MB-231 cells are shown below. Figure 3 As shown, column 1 shows the fluorescence distribution of compounds 4, 5, 9, and 10 in MDA-MB-231 cells, with green fluorescence representing the corresponding compounds; column 3 shows real MDA-MB-231 cell images under a laser confocal microscope; column 2 shows fluorescence images in real MDA-MB-231 cells. These results collectively demonstrate that the novel fluorescent compounds designed and synthesized in this study can effectively trace their distribution in cells and can serve as molecular tool drugs for further research on anti-tumor mechanisms of action.

[0131] The legend is explained below:

[0132] The scale in the diagram is 20mm.

[0133] Example 13: Duration of action of the compound in MDA-MB-231 cells

[0134] 1. Experimental Methods:

[0135] ① Cell plating: Adherent MDA-MB-231 cells in logarithmic growth phase and in good condition were digested and counted at a concentration of 1.5 × 10⁻⁶. 4 / wells were seeded in 35mm laser confocal culture dishes and cultured in a 37℃, 5% CO2 cell culture incubator;

[0136] ② Cell drug delivery: After cells adhered to the cell plate for 24 hours, 40 μM of covalent fluorescent compound and non-covalent fluorescent compound were added respectively. After incubation in a cell culture incubator for 8 hours, the cells were washed 3 times with PBS and then cultured in the incubator for another 8 hours.

[0137] The covalent fluorescent compound is compound 4-12, and the non-covalent compound is compound 1 in patent CN111423438A and the preferred compound H1k in the literature (Eur.J.Med.Chem.2023,250,115193).

[0138] ③ Microscopic fluorescence observation: At time points of 0, 0.5, 1, 2, 4 and 8 hours of continued culture, observations and images were taken under a laser confocal microscope. The excitation wavelength was 405 nm and the emission wavelength was 465 nm.

[0139] 2. Experimental results: Under laser confocal microscopy, it was observed that the fluorescence of non-covalent compounds decreased rapidly over time, while the fluorescence of covalent compounds remained persistent. This result indicates that covalent compounds can bind to the target site persistently, are not easily expelled by cells in vivo, and can exhibit better and longer-term anti-proliferative activity.

[0140] The above description is only a partial embodiment of this application and an explanation of the technical principles used.

[0141] Those skilled in the art should understand that the scope of the invention involved in this application is not limited to the technical solutions formed by the combination of the above-described technical features, but should also cover other technical solutions formed by any combination of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above-described features with, but not limited to, technical features with similar functions disclosed in this application.

Claims

1. Eudistomin Y-type covalent derivatives as shown in formula (I) and their medically acceptable salts, in, R2 is 2,3-epoxypropyl, and R1 is methyl, ethyl, n-butyl, cyclopropylmethyl, cyclohexylmethyl, (tetrahydropyran-4-yl)methyl or 4-methoxybenzyl. Alternatively, R2 may be acetonitrile, and R1 may be cyclohexylmethyl or 4-methoxybenzyl.

2. The Eudistomin Y-type covalent derivative of formula (I) as described in claim 1 and its medically acceptable salt, characterized in that... The Eudistomin Y-type covalent derivative shown in formula (I) is one of the following chemical formulas:

3. The method for preparing Eudistomin Y-type covalent derivatives as described in claim 1, characterized in that... The method includes the following steps: (1) Using tryptophan methyl ester as a raw material, it is reacted with 4-methoxyacetophenone, I2 and H2O2 in a molar ratio of 1:1~1.5:0.8~2:1.5~2.5 to obtain the intermediate compound shown in Formula 1; (2) The intermediate compound shown in Formula 1 is reacted with R1-X and K2CO3 in a molar ratio of 1:1.7 to 6:1.2 to 7 to obtain the compound shown in Formula 2; the X group in R1-X is a halogen element; (3) The compound shown in Formula 2 is hydrolyzed under alkaline conditions to obtain the compound shown in Formula 3; (4) The compound shown in Formula 3 was reacted with R2-Y and K2CO3 in a molar ratio of 1:1.2 to 10:1.5 to 8 to prepare Eudistomin Y-type covalent derivatives as shown in Formula (I); the Y group in R2-Y is a halogen element; The reaction formula for the method is as follows:

4. The method as described in claim 3, characterized in that... The reaction conditions for step (3) are an alkaline environment with a pH value of 8-13.

5. The use of Eudistomin Y covalent derivatives of formula (I) as described in claim 1 or 2 and their medically acceptable salts in the preparation of antitumor drugs.

6. The application as described in claim 5, characterized in that... The anti-tumor drug mentioned is a drug for treating triple-negative breast cancer.

7. The application as described in claim 5, characterized in that... The antitumor drug is a long-acting antitumor drug.

8. The use of Eudistomin Y covalent derivatives as described in claim 1 or 2 and their medically acceptable salts in the preparation of antitumor tracer molecular tool drugs.

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